Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions

Article abstract of DOI:10.1021/jacs.5b09817

Synthetic agents that disrupt intracellular protein-protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional

Full text of DOI:10.1021/jacs.5b09817

Communication
pubs.acs.org/JACS
Intracellular Generation of a Diterpene-Peptide Conjugate that
Inhibits 14-3-3-Mediated Interactions
†
‡
†
†
,†
Prakash Parvatkar, Nobuo Kato, Motonari Uesugi, Shin-ichi Sato, and Junko Ohkanda*
†Institute for Chemical Research and Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Gokasho, Uji,
Kyoto 611-0011, Japan
‡
Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
*
S Supporting Information
The 14-3-3 proteins are a family of dimeric proteins that are
expressed in all eukaryotic cells and play key regulatory roles in
a diverse array of physiologic processes mediated by serine/
ABSTRACT: Synthetic agents that disrupt intracellular
protein−protein interactions (PPIs) are highly desirable
for elucidating signaling networks and developing new
therapeutics. However, designing cell-penetrating large
molecules equipped with the many functional groups
necessary for binding to large interfaces remains
challenging. Here, we describe a rational strategy for the
intracellular oxime ligation-mediated generation of an
amphipathic bivalent inhibitor composed of a peptide and
diterpene natural product, fusicoccin, which binds 14-3-3
protein with submicromolar affinity. Our results demon-
strate that co-treatment of cells with small module
molecules, the aldehyde-containing fusicoccin 1 and the
aminooxy-containing peptide 2, generates the correspond-
ing conjugate 3 in cells, resulting in significant cytotoxicity.
In contrast, chemically synthesized 3 is not cytotoxic, likely
due to its inability to penetrate cells. Compound 3, but not
6
threonine kinases and phosphatases. Each of the highly helical
monomers possesses a conserved amphipathic groove (∼25 Å
in length) that binds to a consensus phosphopeptide motif
containing either phosphoserine (pS) or phosphothreonine
6b,c
(
pT) residues. Hundreds of intracellular protein ligands have
been identified, including key oncogenic regulators, such as
7
Raf. A number of studies have implicated dysregulation of 14-
8
9
3
1
-3 interactions in cancer and neurologic diseases, suggesting
2,10
4-3-3 as a new therapeutic target.
A previous study conducted by screening a phage display
library identified a 20-mer peptide as a potent 14-3-3
inhibitor. This peptide, R18, binds 14-3-3 with a K of ∼80
nM. R18 occupies the amphipathic groove and thereby
interferes with 14-3-3/native ligand interactions. Moreover,
expression of a dimer form of R18 in cells induces apoptosis,
11
d
1
1
12
1
or 2, disrupts endogenous 14-3-3/cRaf interactions,
thus validating the potential of 14-3-3 as a therapeutic target.
suggesting that cell death is caused by inhibition of 14-3-3
activity. These results suggest that intracellular generation
of large-sized molecules may serve as a new approach for
modulating PPIs.
To date, several hydrophobic small-molecule-based 14-3-3
13
inhibitors have been identified by library screenings, however,
these compounds show moderate activity in cells in the
submillimolar range. The characteristic shallow amphiphilic
binding groove of 14-3-3 increases the difficulty of developing
potent inhibitors.
rotein−protein interactions (PPIs) play crucial roles in
We therefore aimed at developing a strategy to generate large
amphipathic agents based on the intracellular ligation of small
hydrophobic and hydrophilic fragments containing comple-
mentary reactive groups (Figure 1A, red and blue, respectively).
Such intracellularly generated conjugate (Figure 1A, green)
would bind to the large 14-3-3 binding groove and thereby
disrupt 14-3-3 PPIs, enabling the assembly of a nonpermeable
large molecule using smaller cell penetrating fragments.
P
signaling networks that regulate many physiologic
processes. Controlling intracellular PPIs using synthetic agents
1
remains challenging, as dysregulated proteins found in diseases
are often implicated in intracellular signaling circuits (e.g.,
2
kinase/phosphatase-signal transduction). However, the large,
3
featureless, dynamic, and even frequently disordered interfaces
of PPIs limit the development of typical small-molecule-based
cell-penetrating agents, although there are some notable
14
Manetsch et al. reported an in vitro approach in which Bcl-
xL combines thioacids and sulfonylazides into acylsulfonamides.
Screening of the resulting chemical library identified a potent
1
,4
successes. Larger agents are often necessary to target a
correspondingly large surface, and thus, many researchers have
Bcl-X inhibitor, confirming that this target-guided assembly
L
5
15
focused on developing new delivery approaches. We
approach is a robust strategy for PPI-oriented drug discovery.
We wanted to facilitate the in-cell synthesis of PPI inhibitors by
leveraging structure-based design utilizing fragment modules
capable of penetrating cells.
anticipated that we may provide a solution to this obstacle by
delivering two small molecules that would assemble as a larger
molecule through their interaction with a common intracellular
protein target. Here, we describe the rational design of two
small reactive modules based on the structure of a diterpene
natural product and a phosphopeptide for oxime ligation, which
produces a bivalent diterpene-peptide conjugate capable of
disrupting 14-3-3 protein-mediated interactions in cells.
Toward this end, we focused on the diterpene natural
16
product, fusicoccin A (FC, Figure 1B) as the hydrophobic
Received: September 23, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b09817
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
modified FC derivative (1) containing an o-formyl benzyl
group, and peptide 2, in which the carboxy terminus of the
PMA2 peptide was modified to an oxyamino group, was
designed for oxime ligation (Figure 1D). To enhance
membrane penetration and stability in cells, the pT residue
was replaced with aspartic acid.
21
Oxime ligation has been widely used in both in vitro and in
22
vivo studies, as this bioorthogonal condensation produces
stable oxime derivatives under physiologic conditions. More-
23
over, previous studies by Rideout et al. demonstrated that
assembling of bioactive compounds utilizing hydrazone
formation reaction resulted in a synergetic effect on cell-
based activity. Thus, we hypothesized that the two modules
could enter cells and bind 14-3-3, which in turn would template
oxime ligation to yield the amphiphilic diterpene-peptide
conjugate 3 (MW = 1582 Da), which is capable of binding to
1
4-3-3 more tightly than either module alone due to the
24
additive effect of binding energies. Compound 3 was readily
prepared by mixing 1 and 2 in methanol with a catalytic
amount of trifluoroacetic acid (see Supporting Information
[
SI]).
To assess whether the modules were appropriately designed
for 14-3-3-templated ligation, the docking of 1 and 2 to human
4-3-3ζ (see SI for details) was computationally modeled. The
1
model showed that the benzyl group at the 12-position of FC
favorably projects the aldehyde at the ortho position toward the
ammonium group of 2, which forms a hydrogen bond with the
aldehyde oxygen (2.03 Å, Figure 2A), suggesting that the
Figure 1. (A) Schematic of intracellular generation of the 14-3-3
inhibitor (green) by ligation of hydrophobic (red) and hydrophilic
(
blue) modules. (B) Chemical structure of fusicoccin A (FC). (C)
Crystal structure of the ternary complex of 14-3-3, FC, and
17
QSYpTV. (D) Chemical structures of the modules (1, 2) and
conjugate product (3).
module. FC is a phytotoxin produced by the pathogenic fungus
Phomopsis amygdali. FC binds to 14-3-3/ligand complexes and
Figure 2. Docking models of (A) 1 (pale pink stick) and 2 (gray stick,
dansyl-Gly was omitted for calculation), with predicted hydrogen
bonds indicated by red dashed lines, and (B) 3 (green stick with
10c
enhances the interaction, although FC itself is a weak ligand
1
7
for 14-3-3 (K ∼ 66 μM). The reported crystal structure of
d
plant 14-3-3 bound to FC and the PMA2 phosphopeptide
̈
yellow surface) to 14-3-3 (Glide in Schrodinger Suites 2015-2).
(
QSYpTV; the 14-3-3-binding consensus motif found in the C-
+
terminal of plant H -ATPase) clearly revealed that FC binds in
the hydrophobic cavity adjacent to the peptide and forms a
ternary complex (Figure 1C). Notably, formation of this
ternary complex is driven by van der Waals interactions
between FC and the valine residue of the PMA2 peptide,
resulting in an increase in the affinity of both ligands for 14-3-3
of nearly 2 orders of magnitude. This stabilization effect is
thought to account for the toxicity of FC in plants. Moreover,
synthetic analogues of FC have been found to interact with
human 14-3-3 and exhibit unique antitumor activity in
aldehyde and ammonium groups readily react to give the
desired oxime derivative. The model also showed that the
carboxylate of the aspartic acid residue in 2 binds to the
phosphate-binding site of 14-3-3 interacting with lysine49 and
arginine129, validating the mimicry of the phospho group
(Figure 2A). Furthermore, the model indicated that conjugate 3
preserves the original conformation of 1 and 2 with only
minimal changes, suggesting that the oxime ligation does not
cause apparent steric repulsion between 14-3-3 and 3 (Figure
2B). These data suggest that 3 exhibits a complementary
structure and size appropriate for binding to the 14-3-3 groove
(Figure 2B).
17
17
1
8
1
9
animals. These data suggested that FC would be a suitable
motif for development of the proposed amphiphilic bivalent 14-
3-3 inhibitors.
In our modular design strategy, we wanted to maximize the
In vitro evaluations of the reaction of 1 and 2 (300 μM each)
and the template effect of recombinant 14-3-3ζ (300 μM) were
carried out by HPLC in HEPES buffer (Figure 3, see SI for
details). Ligation under 14-3-3-free conditions was found to be
very slow, yielding only a small amount of conjugate 3 by 24 h
(Figure 3, inset, left). In contrast, addition of an equimolar
amount of 14-3-3ζ significantly accelerated the production of 3
(Figure 3, inset, right), with the yield at 1 h more than 50 times
that observed under 14-3-3-free conditions (Figure 3, red and
advantages derived from cooperative formation of the ternary
complex of 14-3-3 bound to FC and the PMA2 peptide. In our
20
earlier study toward 14-3-3-mediated ligation, we attempted
an epoxide-opening reaction using a modified FC derivative
attached to the epoxide group by a long and flexible spacer,
however, the moderate reaction efficiency hampered further
evaluation in cells. Therefore, in order to exploit a proximity
effect for the ligation upon the binding to 14-3-3, a simply
B
DOI: 10.1021/jacs.5b09817
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Journal of the American Chemical Society
Communication
Figure 3. Changes in 2 (300 μM, triangle) and 3 (300 μM, circle) over
time in the absence (white) or presence (red) of 14-3-3ζ (300 μM) in
HEPES (100 mM, pH 7.3), 150 mM NaCl, 0.05% (v/v) Tween 20, 25
Figure 4. (A) HPLC analysis of oxime ligation in cells: (a) lysis buffer,
(b) chemically synthesized 3, (c) lysate of cells treated with DMSO
(0.1% [v/v]), (d) lysate of cells treated with 1 and 2 (50 μM,
°C. Relative peak area = a/(a + b) or b/(a + b), where a and b are the
respectively). (B) Trypan blue assay for HEK293 cells stably
expressing FLAG-14-3-3ζ (n ≥ 3). (C) Western blot of co-
immunoprecipitated proteins using anti-cRaf and -FLAG antibodies.
peak areas of 2 and 3. Insets: HPLC profiles of the reactions in the
absence (left) and presence (right) of 14-3-3.
white circles). The significant difference in reaction rates in the
presence/absence of 14-3-3 is suggestive of a proximity effect
associated with binding to 14-3-3. R18 peptide, a general 14-3-3
inhibitor, reduced the rate of 3 generation by ∼80%, supporting
the hypothesis that the 14-3-3 binding groove is involved in the
ligation reaction (Figure S1).
resulted in dose-dependent cytotoxicity, whereas treatment
with either compound alone resulted in considerably less
cytotoxicity (Figure 4B). Moreover, chemically prepared 3 was
found to have no effect on cell proliferation, even at high
concentrations (Figure 4B). Previous studies revealed that
expression of a dimer form of R18 peptide in cells leads to cell
death. Thus, these results suggest that 3 generated intra-
cellularly binds to and inhibits 14-3-3, resulting in significant
cytotoxicity, whereas chemically synthesized 3 is inactive, most
likely because it cannot penetrate cells due at least in part to its
large molecular size.
In order to evaluate the additive effect of binding energies on
ligation, isothermal titration calorimetry (ITC) of 2 and 3 with
12
25
1
4-3-3ζ was performed. First, we tested the PMA2 peptide
and confirmed its binding to 14-3-3ζ, with a K value of 1.24 ±
d
0
.02 μM (Figure S2A), which was consistent with a previously
17
reported value (K = 2.5 μM). We then evaluated peptide
d
fragment 2 and observed no apparent binding to the protein
Finally, we examined whether 3 can disrupt interactions
between 14-3-3 and its protein ligands. cRaf is an oncogenic
(
Figure S2B), presumably due to the lack of a phosphorylated
group, as a previous study showed that dephosphorylation of a
4-3-3 ligand peptide, SWpTY (K = 0.17 μM), significantly
7
kinase that is regulated by 14-3-3 and is a well-studied
27
1
potential target for cancer therapeutics. Thus, we conducted a
co-immunoprecipitation experiment with FLAG-14-3-3ζ to
examine the effect of the compounds on the binding of cRaf to
14-3-3. Lysates of HEK293 cells expressing FLAG-14-3-3ζ were
treated with anti-FLAG antibody affinity beads in the presence
of test compounds (20 μM), and bound proteins were eluted
using 3× FLAG peptide and analyzed by Western blotting
using anti-cRaf and -FLAG antibodies. No significant differ-
ences in the cRaf bands were observed between the DMSO
control and 1 or 2, whereas 3 significantly reduced the intensity
of the cRaf band (Figure 4C), demonstrating that the conjugate
3, but not 1 or 2, indeed disrupts the endogenous cRaf/14-3-3
interaction. As 3 was designed to occupy the entire binding
groove of 14-3-3, this compound most likely acts as a general
inhibitor, disrupting 14-3-3 interactions globally, which would
account for its significant cytotoxicity.
d
reduces its affinity, by more than 600-fold (SWTY, K > 100
d
2
6
μM). In contrast, the conjugate 3 exhibited clear 1:1 binding
to 14-3-3ζ, with a K value of 0.37 ± 0.14 μM (ΔG = −1.96,
d
ΔH = −1.33, −TΔS = 0.63 kcal/mol; Figure S2C), an affinity
even greater than that of PMA2. This submicromolar affinity of
the conjugate 3 was further confirmed by the competitive
fluorescence anisotropy titration (K = 0.62 ± 0.35 μM; for
d
more details, see SI). These results demonstrate that attach-
ment of the diterpene moiety to 2 converts it to an amphiphilic
compound, remarkably improving affinity due to the bivalency
introduced by the covalent linking of 1 and 2.
In order to test whether the ligation reaction could be carried
out intracellularly, we prepared HEK293 cells stably expressing
5
FLAG-14-3-3ζ. Cells (2.5 × 10 ) were treated with 1 and 2 (50
μM, 25 nmol, each), incubated for 12 h, and then washed with
medium (3×) and buffer (4×). A cell lysate was obtained,
lyophilized, extracted with methanol, and then analyzed by
HPLC (for more details, see SI). The HPLC results clearly
showed a peak corresponding to 3, indicating that the
conjugate was generated in the cells (Figure 4A). The amount
of 3 generated was estimated at ∼11 nmol (yield ∼45%; for
more details, see SI), indicating that the oxime ligation
proceeded to produce 3 at a decent yield. The potential
limitation of the cell permeation of 2 could be one of the reason
for the moderate yield. Furthermore, treatment of cells with an
equimolar mixture of 1 and 2 for a longer period (24 h)
In conclusion, this work presents a new approach for
generating 14-3-3 inhibitors by oxime ligation between
diterpene and peptide modules that are designed based on
the co-crystal structure of 14-3-3. We demonstrated intra-
cellular generation of 3, which induces cell death, presumably
by inhibiting 14-3-3 PPIs. Importantly, chemically synthesized
3 does not induce cytotoxicity, likely due to its inability to
penetrate cells, validating the strategy of constructing large,
nonpenetrating agents within cells as an alternative means of
modulating the 14-3-3 signaling network. To the best of our
knowledge, the diterpene conjugate 3 is one of the strongest
C
DOI: 10.1021/jacs.5b09817
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
nonpeptidic ligands for 14-3-3 reported to date. Therefore, this
work also suggests that the series of fusicoccins hold a promise
as scaffolds for further development of inhibitors of 14-3-3,
which has received intense research attention as a potential new
drug target for treating cancers and neurodegenerative diseases.
In humans, there are seven isoforms of 14-3-3 proteins with a
highly preserved amphipathic binding groove. A potential
limitation of our 14-3-3 inhibitor is that it may likely inhibit all
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b09817.
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AUTHOR INFORMATION
Corresponding Author
johkanda@scl.kyoto-u.ac.jp
■
*
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Prof. Michael Kahn (University of Southern
California) for providing the 14-3-3 plasmid. This work was
supported by the JSPS (25·3344 to P.P., and 25288076,
6102727 to J.O.); the Cooperative Research Program,
Network Joint Research Center for Materials and Devices,
Osaka University (2015349 to J.O.); and in part by JSPS grants
(
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rtele, M.; Ottmann, C. J.; Wittinghofer, A.; Oecking, C.
2
(
2
6220206 (to M.U.) and 23510277 (to S.S.). iCeMS is
(
supported by the World Premier International Research Center
Initiative (WPI), MEXT, Japan. This work was inspired by the
international and interdisciplinary environments of the iCeMS
and JSPS Asian CORE Program, “Asian Chemical Biology
Initiative.” J.O. sincerely thanks Prof. Indraneel Ghosh
J.; Kato, N.; Tongu, M.; Yamada, T.; Akimoto, M.; Takenaga, K.;
Sassa, T.; Suzumiya, J.; Honma, Y. Anti-Cancer Agents Med. Chem.
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(
University of Arizona) for critical reading of the manuscript
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Wagner, C. R.; Distefano, M. D. J. Am. Chem. Soc. 2013, 135, 16388.
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX